Comprehensive virome profiling of sugarcane and simplified duplex OneStep RT-PCR assay reveals the prevalence of sugarcane streak mosaic virus along with sugarcane yellow leaf virus in India

IF 3.5 Q3 Biochemistry, Genetics and Molecular Biology

Journal of Genetic Engineering and Biotechnology Pub Date : 2024-11-26 DOI:10.1016/j.jgeb.2024.100442

Nishant Srivastava , Malyaj R. Prajapati , Rakesh Kumar , Pooja Bhardwaj , Nitika Gupta , Vanita Chandel , Susheel K. Sharma , Virendra K. Baranwal

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Abstract

Background

Sugarcane is host of many viral pathogens that affects its growth and productivity. High-throughput sequencing (HTS) is comprehensive diagnostic platform that permit the precise detection of viral pathogens to resolve the disease epidemiology of the crop, thus providing the phytosanitary status of plants. The current work was designed to comprehend the virome profiling of sugarcane belonging to five varieties collected from the major crop producing states in India. Additionally, a duplex OneStep RT-PCR assay was optimized for simplified detection of prevalent viruses in single reaction run along with validation and confirmation of HTS results.

Results

The complete genome sequences of sugarcane streak mosaic virus (SCSMV), sugarcane yellow leaf virus (SCYLV) and sugarcane mosaic virus (SCMV) consisted of 9790, 5849 and 9600 nucleotides (nt) respectively were obtained excluding 5′ UTR and 3′ poly (A) tail from sugarcane samples belonging to different varieties. SCSMV and SCMV had single ORF encoding 3130 and 3063 amino acids (aa) respectively, whereas SCYLV genome comprised of six ORFs. The proteolytic cleavage sites in polyprotein region of SCSMV and SCMV revealed the unique amino acid motifs. SCSMV generated the highest number of single nucleotide variants (SNVs) 876 suggesting that it is more susceptible to mutations than other elucidated viruses in HTS. Recombination events revealed the origin of SCSMV_UP isolate from Indian and Iranian isolates as major and minor parents respectively. Further, validation assay by simplified duplex OneStep RT-PCR revealed the prevalence of SCSMV and SCYLV as mixed infection in sugarcane samples with 28 % incidence. The assay could detect the viruses up to 100 pg/µL of RNA concentration.

Conclusion

The first comprehensive report of sugarcane virome and use of an optimized duplex OneStep RT-PCR assay revealed the prevalence of SCSMV and SCYLV in sugarcane from India. The study also provides an insight into genetic variations in the coding region of SCSMV and SCMV and emergence of diverse variants present in a viral population. A simplified duplex OneStep RT-PCR assay for simultaneous and expeditious detection of prevalent viruses in sugarcane would be useful in certification programme for production of virus-free planting materials.

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甘蔗病毒组综合分析和简化的双联 OneStep RT-PCR 检测揭示了甘蔗条纹花叶病毒和甘蔗黄叶病毒在印度的流行情况

背景甘蔗是多种病毒病原体的宿主，这些病原体会影响甘蔗的生长和产量。高通量测序（HTS）是一种综合诊断平台，可精确检测病毒病原体，从而了解作物的疾病流行情况，并提供植物检疫状况。目前的工作旨在了解从印度主要作物生产邦收集的五个甘蔗品种的病毒组图谱。此外，还优化了双联一步 RT-PCR 分析法，以简化单次反应中流行病毒的检测，同时验证和确认 HTS 结果。结果从不同品种的甘蔗样本中获得了甘蔗条纹花叶病毒（SCSMV）、甘蔗黄花叶病毒（SCYLV）和甘蔗花叶病毒（SCMV）的完整基因组序列，分别包含 9790、5849 和 9600 个核苷酸（nt），其中不包括 5′ UTR 和 3′ poly (A) 尾。SCSMV 和 SCMV 的单 ORF 分别编码 3130 和 3063 个氨基酸（aa），而 SCYLV 基因组由 6 个 ORF 组成。SCSMV 和 SCMV 多蛋白区的蛋白水解位点显示了独特的氨基酸基序。SCSMV产生的单核苷酸变体（SNV）数量最多，达876个，表明它比其他已阐明的HTS病毒更容易发生突变。重组事件表明，SCSMV_UP 分离物的主要亲本和次要亲本分别来自印度和伊朗分离物。此外，通过简化的双工一步法 RT-PCR 验证检测发现，甘蔗样本中的 SCSMV 和 SCYLV 混合感染率为 28%。结论首次全面报告甘蔗病毒组并使用优化的双工一步法 RT-PCR 检测方法揭示了 SCSMV 和 SCYLV 在印度甘蔗中的流行情况。该研究还揭示了 SCSMV 和 SCMV 编码区的遗传变异以及病毒群体中出现的多种变体。用于同时快速检测甘蔗中流行病毒的简化双联 OneStep RT-PCR 检测方法将有助于无病毒种植材料生产认证计划。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Journal of Genetic Engineering and Biotechnology Biochemistry, Genetics and Molecular Biology-Biotechnology

CiteScore

5.70

自引率

5.70%

发文量

159

审稿时长

16 weeks

期刊介绍： Journal of genetic engineering and biotechnology is devoted to rapid publication of full-length research papers that leads to significant contribution in advancing knowledge in genetic engineering and biotechnology and provide novel perspectives in this research area. JGEB includes all major themes related to genetic engineering and recombinant DNA. The area of interest of JGEB includes but not restricted to: •Plant genetics •Animal genetics •Bacterial enzymes •Agricultural Biotechnology, •Biochemistry, •Biophysics, •Bioinformatics, •Environmental Biotechnology, •Industrial Biotechnology, •Microbial biotechnology, •Medical Biotechnology, •Bioenergy, Biosafety, •Biosecurity, •Bioethics, •GMOS, •Genomic, •Proteomic JGEB accepts